Primary immunodeficiency; autoimmunity; exome sequencing; deletion; gene; CVID
The cause of corticosteroid resistant asthma is unknown.
To perform gene microarray analyses using BAL cells from well-characterized corticosteroid resistant (CR) and sensitive (CS) asthmatics to elucidate the differential expression of genes that contribute to the development of corticosteroid resistance.
The patients were characterized as CR or CS based on FEV1% predicted improvement after one week course of oral prednisone. Expression of selected gene targets was verified by real time PCR and by ELISA.
Microarray analyses demonstrated significantly higher levels (over three-fold increase, p<0.05) of transcripts for TNFα, IL-1α, IL-1β, IL-6, CXCL1, CXCL2, CXCL3, CXCL8 (IL-8), CCL3, CCL4, CCL20 in BAL cells of CR asthmatics. These findings, confirmed by RT-PCR in additional BAL samples, were consistent with classical macrophage activation by bacterial products. In contrast, markers of alternatively-activated macrophages, Arginase I and CCL24, were decreased. Genes associated with activation of the LPS signaling pathway (EGR1, DUSP2, MAIL, TNFAIP3) were significantly elevated in CR BAL samples (p<0.05). These patients had significantly higher amounts (1444.0±457.3 pg per mg of total protein) of LPS in BAL fluid than CS asthmatics (270.5±216.0 pg; p<0.05) as detected by LAL assay and confirmed by gas chromatography mass spectrometry analysis. Pronged exposure to LPS induced functional steroid resistance to dexamethasone (DEX) in normal monocytes, demonstrated by persistently elevated IL-6 levels in the presence of DEX.
Classical macrophage activation and induction of LPS signaling pathways along with high endotoxin levels detected in BAL fluid from CR asthmatics suggest that LPS exposure may contribute to CR asthma.
corticosteroids; asthma; resistance; genes; endotoxin
Viral respiratory infections can have a profound effect on many aspects of asthma including its inception, exacerbations, and, possibly, severity. Of the many viral respiratory infections that influence asthma, the common cold virus, rhinovirus, has emerged as the most frequent illness associated with exacerbations and other aspects of asthma. The mechanisms by which rhinovirus influences asthma are not fully established, but current evidence indicates that the immune response to this virus is critical in this process. Many airway cell types are involved in the immune response to rhinovirus, but most important are respiratory epithelial cells and possibly macrophages. Infection of epithelial cells generates a variety of proinflammatory mediators to attract inflammatory cells to the airway with a subsequent worsening of underlying disease. Furthermore, there is evidence that the epithelial airway antiviral response to rhinovirus may be defective in asthma. Therefore, understanding the immune response to rhinovirus is a key step in defining mechanisms of asthma, exacerbations, and, perhaps most importantly, improved treatment.
Asthma; exacerbation; rhinovirus; immune response to virus; pathogenesis
IL-13 is a central mediator of airway responsiveness and mucus expression in allergic airway inflammation and IL-13 is currently a therapeutic target for asthma. However, little is known about how IL-13 regulates human CD4+ T cell lineages because the IL-13 receptor α1 (IL-13Rα1), a subunit of the IL-13 receptor, has not previously been reported to exist on human T cells.
To determine if human CD4+ Th17 cells express IL-13Rα1 and if IL-13 regulates Th17 cytokine production.
Naïve human CD4+ cells were isolated from whole blood, activated with anti-CD3 and anti-CD28, and polarized to Th1, Th2, Th17, or induced T regulatory cells in the presence of IL-13 (0–10ng/ml). Cell supernatants, total RNA, or total protein was examined four days after Th17 polarization.
Th17 cells, but not Th0, Th1, Th2 or induced T regulatory cells, expressed IL-13Rα1. IL-13 attenuated IL-17A production as well as expression of RORC2, Runx1, and IRF-4 in Th17 polarized cells. IL-13 neither inhibited IFN-γ production from Th1 cells nor inhibited IL-4 production from Th2 cells. Furthermore, attenuation of IL-17A production only occurred when IL-13 was present within 24 hours of T cell activation or at the time of restimulation.
IL-13Rα1 is expressed on human CD4+ Th17 cells, and IL-13 attenuates IL-17A production at polarization and restimulation. While IL-13 is an attractive therapeutic target for decreasing symptoms associated with asthma, these results suggest that therapies inhibiting IL-13 production could have adverse side effects by increasing IL-17A production.
IL-13; IL-13R; Th17; IL-17A; Asthma
Interleukin-4 (IL-4) and STAT6 play an important role in progression of allergic airway disease (AAD) or asthma. IL-4 and STAT6 mediate T helper 2 (Th2) responses in T cells, and immunoglobulin class switching to IgE in B cells. Both, Th2 responses and IgE promote the asthmatic condition. We have previously demonstrated that PARP-14, a member of the poly ADP-ribose polymerase (PARP) family of proteins regulates the transcription function of STAT6. However, the role of PARP-14 in AAD is not known.
Here we investigate the role of PARP-14 and the enzyme activity associated with it in AAD dependent on airway hyper-responsiveness (AHR) and lung inflammation. We also elucidate the mechanism by which PARP-14 regulates AAD.
The role of PARP-14 and its enzyme activity in AAD and Th2 differentiation were examined using a mouse model of AAD and in vitro T helper cell differentiation.
PARP-14 deficient animals when compared to controls show reduced lung pathology and IgE. Treating mice with a pharmacological inhibitor for PARP activity reduced the severity of AHR and lung inflammation. Mechanistically, our data indicate that PARP-14 and its enzyme activity aid in the differentiation of T cells towards a Th2 phenotype by regulating the binding of STAT6 to the Gata3 promoter.
PARP-14 and the catalytic activity associated with it promote Th2 differentiation and AAD in a murine model, and targeting PARP-14 may be a potential new therapy for allergic asthma.
Interleukin-4; STAT6; PARP-14; Th2 cells; Gata3; lung inflammation; airway hyper-responsiveness; PARP inhibitor; allergic airway disease
GSTM1null genotype; PMN responsiveness; ozone; innate immune phenotypes
Whereas rodent studies indicate that atherosclerosis is a T helper (Th)1-mediated disease and that atopic Th2 immunity is atheroprotective, findings in humans are conflicting. Total IgE (tIgE) is associated with atherosclerotic disease, but has limited specificity for atopy.
Our aim was to determine the relationship between atopy, as indicated by a broad panel of serum allergen-specific immunoglobulin E (sIgE), and past myocardial infarction (MI) in a sample representative of the U.S. population.
Data were analyzed from 4,002 participants aged ≥20 years from the 2005–2006 National Health and Nutrition Examination Survey.
Subjects reporting a history of MI had lower summed sIgE (5.51 vs. 7.71 kU/L; P<0.001), and were less likely to have ≥1 positive sIgE test (29.9% vs. 44.6%; P=0.02) or current hay fever (3.3% vs. 7.6%; P=0.002). After adjustment for age, gender, race/ethnicity, diabetes mellitus, hypertension, family history of MI, smoking, total/high density lipoprotein-cholesterol, body mass index, and C-reactive protein, the odds ratio (OR) for MI was 0.91 (95% confidence interval [CI], 0.85–0.97) per positive sIgE; 0.70 (95% CI, 0.57–0.85) per 2-fold increase in sum[sIgE]; and 0.82 (95% CI, 0.69–0.98) per 10% increase in the ratio of sum[sIgE] to tIgE. Analysis using 7 data-driven, prespecified allergen clusters revealed that house dust mite is the only allergen cluster for which sIgE is associated with reduced odds for MI (fully adjusted OR 0.36 [95% CI, 0.20–0.64]).
Serum sIgE is inversely related to MI in the U.S. population in a manner independent of multiple coronary risk factors.
Myocardial Infarction; Immune System; Risk Factors; Atopy; Immunoglobulin E
Hypereosinophilic syndromes (HES) are chronic disorders that require long-term therapy to suppress eosinophilia and clinical manifestations. Corticosteroids are usually effective, yet many patients become corticosteroid-refractory or develop corticosteroid toxicity. Mepolizumab, a humanised monoclonal anti-interleukin-5 antibody, demonstrated corticosteroid-sparing effects in a double-blind, placebo-controlled study of FIP1L1/PDGFRA-negative, corticosteroid-responsive subjects with HES.
To evaluate long-term safety and efficacy of mepolizumab (750 mg) in HES.
MHE100901 is an open-label extension study. The primary endpoint was the frequency of adverse events (AEs). Optimal dosing frequency, corticosteroid-sparing effect of mepolizumab, and development of anti-mepolizumab antibodies were also explored.
Seventy-eight subjects received 1–66 mepolizumab infusions each (including mepolizumab infusions received in the placebo-controlled trial). Mean exposure was 251 weeks (range 4–302). The most common dosing interval was 9–12 weeks. The incidence of AEs was 932 events per 100 subject-years in the first year, declining to 461 events per 100 subject-years after 48 months. Serious AEs, including one death, were reported by the investigator as possibly due to mepolizumab in three subjects. The median daily prednisone dose decreased from 20.0 to 0 mg in the first 24 weeks. The median average daily dose for all subjects over the course of the study was 1.8 mg. Sixty-two percent of subjects were prednisone-free without other HES medications for ≥12 consecutive weeks. No neutralizing antibodies were detected. Twenty-four subjects withdrew prior to study completion for death (n=4), lack of efficacy (n=6), or other reasons.
Mepolizumab was well tolerated and effective as a long-term corticosteroid-sparing agent in PDGFRA-negative HES.
eosinophil; monoclonal antibody; interleukin-5; corticosteroid
Wiskott–Aldrich syndrome; Immunoglobulin A
DNA methylation; respiratory hypersensitivity; saliva; pyrosequencing; wheezing; traffic-related air pollution
Sphingosine-1-phosphate (S1P) produced by two sphingosine kinase isoenzymes, SphK1 and SphK2, has been implicated in IgE-mediated mast cell responses. However, studies of allergic inflammation in isotype-specific SphK knockout mice have not clarified their contribution and the role that S1P plays in vivo in a mast cell and IgE-dependent mouse model of allergic asthma has not yet been examined.
We used an isoenzyme-specific SphK1 inhibitor, SK1-I, to investigate the contributions of S1P and SphK1 to mast cell dependent airway hyperresponsiveness (AHR) and airway inflammation in mice.
Allergic airway inflammation and AHR were examined in a mast cell-dependent mouse model of ovalbumin (OVA)-induced asthma. C57BL/6 mice received intranasal delivery of SK1-I prior to sensitization and challenge with OVA or only prior to challenge.
SK1-I inhibited antigen-dependent activation of human and murine mast cells and suppressed activation of NF-κB, a master transcription factor that regulates expression of pro-inflammatory cytokines. SK1-I treatment of mice sensitized to OVA in the absence of adjuvant, which develop mast cell-dependent allergic inflammation, significantly reduced OVA-induced AHR to methacholine; decreased numbers of eosinophils and levels of the cytokines IL-4, 5, 6, 13, IFN-γ, and TNF-α and the chemokines eotaxin, and CCL2 in bronchoalveolar lavage fluid; and decreased pulmonary inflammation as well as activation of NF-κB in the lungs.
S1P and SphK1 play important roles in mast cell-dependent, OVA-induced allergic inflammation and AHR, in part by regulating the NF-κB pathway.
sphingosine-1-phosphate; sphingosine kinase; mast cells; NF-kB; airway hyperresponsiveness; asthma
The prevalence of peanut allergies is rising. Peanuts and many other allergen sources contain significant amounts of triglycerides, which affect absorption of antigens but have unknown effects on sensitization and anaphylaxis. We recently reported that dietary medium-chain triglycerides (MCT), which bypass mesenteric lymph and directly enter portal blood, reduce intestinal antigen absorption into blood compared to long-chain triglycerides (LCT), which stimulate mesenteric lymph flow and are absorbed in chylomicrons via mesenteric lymph.
Test how dietary MCT affect food allergy.
C3H/HeJ mice were fed peanut butter protein in MCT, LCT (peanut oil), or LCT plus an inhibitor of chylomicron formation (Pluronic L81; “PL81”). Peanut-specific antibodies in plasma, responses of the mice to antigen challenges, and intestinal epithelial cytokine expression were subsequently measured.
MCT suppressed antigen absorption into blood, but stimulated absorption into Peyer's patches. A single gavage of peanut protein with MCT as well as prolonged feeding in MCT-based diets caused spontaneous allergic sensitization. MCT-sensitized mice experienced IgG-dependent anaphylaxis upon systemic challenge and IgE-dependent anaphylaxis upon oral challenge. MCT feeding stimulated jejunal-epithelial TSLP, IL-25 and IL-33 expression compared to LCT, and promoted Th2 cytokine responses in splenocytes. Moreover, oral challenges of sensitized mice with antigen in MCT significantly aggravated anaphylaxis compared to challenges with LCT. Importantly, effects of MCT could be mimicked by adding PL81 to LCT, and in vitro assays indicated that chylomicrons prevent basophil activation.
Dietary MCT promote allergic sensitization and anaphylaxis by affecting antigen absorption and availability and by stimulating Th2 responses.
Peanut allergy; triglycerides; TSLP; adjuvant; chylomicrons; intestinal epithelium; Th2 responses
Magnetic resonance imaging (MRI) with 3He does not require ionizing radiation and has been shown to detect regional abnormalities in lung ventilation and structure in adult asthma, but the method has not been extended to childhood asthma. Measurements of regional lung ventilation and microstructure in childhood asthma could advance our understanding of disease mechanisms.
To determine whether 3He MRI in children can identify abnormalities related to diagnosis of asthma or prior history of respiratory illness.
Forty-four children aged 9-10 years were recruited from a birth cohort at increased risk of developing asthma and allergic diseases. For each subject a time-resolved three-dimensional (3D) image series and a 3D diffusion-weighted image were acquired in separate breathing maneuvers. The number and size of ventilation defects were scored, and regional maps and statistics of average 3He diffusion length were calculated.
Children with mild to moderate asthma had lower average diffusion length,
Xrms¯ (p=0.004), increased regional standard deviation of diffusion length (p=0.03), and higher defect scores (p=0.03) than those without asthma. Children with histories of wheezing illness with rhinovirus infection prior to the third birthday had lower
Xrms¯ (p=0.01) and higher defect score (p=0.05).
MRI with 3He detected more and larger regions of ventilation defect and a greater degree of restricted gas diffusion in children with asthma compared to those without asthma. These measures are consistent with regional obstruction and smaller and more regionally variable dimensions of the peripheral airways and alveolar spaces.
asthma; pediatric; hyperpolarized MRI; apparent diffusion coefficient
IgE cross-linking triggers many cellular processes that drive allergic disease. While the role of IgE in mediating allergic responses is best described on basophils and mast cells, expression of the high-affinity IgE receptor on other innate immune cells, including monocytes, suggests that it may impact the function of these cells in allergic environments.
To determine the effect of IgE cross-linking on the function of human monocytes.
Monocytes purified from healthy donor blood samples were cultured for 4–96 hr with media alone, a cross-linking anti-IgE antibody, or control IgG. Surface CD14 and CD64 expression and secreted cytokine concentrations were determined. Monocyte function was determined by assessing: 1) phagocytosis of E. coli or apoptotic HEp2 cells and 2) killing of intracellular E. coli. Select experiments were performed on monocytes obtained from participants with elevated versus normal serum IgE concentrations.
IgE cross-linking on monocytes increased CD14 expression and induced secretion of TNF-á, IL-6, and autoregulatory IL-10. These effects were greatest in individuals with elevated serum IgE concentrations. In contrast, IgE cross-linking reduced CD64 expression and significantly impaired phagocytic function without disrupting the capacity of monocytes to kill bacteria.
IgE cross-linking drives monocyte pro-inflammatory processes and autoregulatory IL-10 in a serum IgE-dependent manner. In contrast, monocyte phagocytic function is critically impaired by IgE cross-linking. Our findings suggest that IgE cross-linking on monocytes may contribute to allergic disease by both enhancing detrimental inflammatory responses and concomitantly crippling phagocytosis, a primary mechanism utilized by these cells to resolve inflammation.
Monocyte; IgE; FcεRI; IgE cross-linking; Allergy; Pro-inflammatory; Autoregulatory; Phagocytosis; Apoptotic debris
The potential consequences of asthma in childhood and young adulthood on lung structure in older adults have not been studied in a large, population-based cohort.
The authors hypothesized that a history of asthma onset in childhood (age 18 or before) or young adulthood (age 19 to 45) was associated with altered lung structure on computed tomography (CT) in later life.
The Multi-Ethnic Study of Atherosclerosis Lung Study recruited 3,965 participants and assessed asthma history using standardized questionnaires, spirometry following guidelines, and segmental airway dimensions and percent low attenuation areas on CT scans.
Asthma with onset in childhood and young adulthood was associated with large decrements in the forced expiratory volume in one second among participants with a mean age of 66 years (−365 ml and −343 ml, respectively; P<0.001). Asthma with onset in childhood and young adulthood was associated with increased mean airway wall thickness standardized to an internal perimeter of 10 mm (Pi10) (0.1 mm, P<0.001 for both), predominantly from narrower segmental airway lumens (−0.39 mm and −0.34 mm, respectively; P<0.001). Asthma with onset in childhood and young adulthood also was associated with a greater percentage of low attenuation areas (1.69% and 4.30%, respectively; P<0.001). Findings were similar among never smokers except that differential percentage of low attenuation areas in child-onset asthma was not seen in them.
Asthma with onset in childhood or young adulthood, was associated with reduced lung function, narrower airways and, among asthmatics who smoked, greater percentage of low attenuation areas in later life.
airway remodeling; airway structure; asthma; emphysema; epidemiology
Recent data, primarily from Europe, suggest children with atopic dermatitis may be at increased risk of developing mental health disorders.
We aimed to quantify the mental health burden associated with pediatric atopic dermatitis in the United States.
A cross-sectional study design was used analyzing data from the 2007 National Survey of Children's Health – a survey reporting on the health status of 92,642 non-institutionalized children ages 0-17. The lifetime prevalence of various provider-diagnosed mental health conditions was calculated for those with and without a history of atopic dermatitis.
The odds of having attention-deficit/hyperactivity disorder was significantly increased in children with atopic dermatitis compared to non-atopic dermatitis controls, OR 1.87 (95% CI 1.54, 2.27) even after controlling for known confounders. The adjusted odds ratios for depression, anxiety, conduct disorder, and autism were 1.81 (95% CI 1.33,2.46) , 1.77 (95% CI 1.36, 2.29), 1.87 (1.46, 2.39), and 3.04 (95% CI 2.13, 4.34), respectively, and these estimates were all statistically significant. A clear dose-dependent relationship was observed between the prevalence of a mental health disorder and the reported severity of the skin disease.
Our data reveal a striking association between mental health disorders and atopic dermatitis in the U.S. pediatric population. The severity of the skin disease alters the strength of the association. Prospective cohort studies are needed to verify these associations and to explore underlying mechanisms. Strategies to prevent atopic dermatitis or to aggressively treat early skin inflammation may modify the risk of developing mental health disorders in at-risk children.
Atopic dermatitis; Comorbidities; Attention-deficit/hyperactivity disorder; Anxiety; Depression; Autism; Prevalence
next generation sequencing; FOXP3; IPEX; infections; food allergy
Sensitization to food antigen may occur through cutaneous exposure.
Test the hypothesis that epicutaneous (EC) sensitization with food antigen predisposes to IgE-mediated anaphylaxis upon oral allergen challenge.
BALB/c mice were EC sensitized by repeated application of ovalbumin (OVA) to tape-stripped skin over 7 weeks, or orally immunized with OVA and cholera toxin (CT) weekly for 8 weeks, then orally challenged with OVA. Body temperature was monitored and serum mouse mast cell protease 1 (mMCP-1) level was determined following challenge. Tissue mast cells (MCs) were examined by chloroacetate esterase (CAE) staining. Serum OVA-specific IgE and IgG1 antibodies, and cytokines in supernatants of OVA-stimulated splenocytes, were measured by ELISA. Serum interleukin-4 (IL-4) levels were measured using an in vivo cytokine capture assay (IVCCA).
EC sensitized mice exhibited expansion of connective tissue MC in the jejunum, increased serum IL-4 levels, and systemic anaphylaxis following oral challenge, as evidenced by decreased body temperature and increased serum mMCP-1 level. Intestinal MC expansion and anaphylaxis were IgE-dependent, as they did not occur in EC sensitized IgE−/− mice. Mice orally immunized with OVA+CT failed to increase serum IL-4 levels, expand their intestinal MCs, or develop anaphylaxis following oral challenge, despite OVA-specific IgE levels and splenocyte cytokine production in response to OVA stimulation, which were comparable to those of EC sensitized mice.
EC sensitized mice, but not mice orally immunized with antigen+CT, develop expansion of intestinal MCs and IgE-mediated anaphylaxis following single oral antigen challenge. IgE is necessary but not sufficient for food anaphylaxis, and MC expansion in the gut may play an important role in the development of anaphylaxis.
The skin may be an important route of sensitization to food antigens. Avoidance of cutaneous sensitization may prevent the development of food anaphylaxis.
Food allergy; epicutaneous sensitization; IgE; mast cells; anaphylaxis
Mast cells express receptors for complement anaphylatoxins C3a and C5a (i.e., C3aR and C5aR), and C3a and C5a are generated during various IgE-dependent immediate hypersensitivity reactions in vivo. However, it is not clear to what extent mast cell expression of C3aR or C5aR influences C3a- or C5a-induced cutaneous responses or IgE-dependent mast cell activation and passive cutaneous anaphylaxis (PCA), in vivo.
Assess whether mouse skin mast cell expression of C3aR or C5aR influences: 1) the cells’ responsiveness to intra-dermal injections of C3a or C5a, or 2) the extent of IgE-dependent mast cell degranulation and PCA in vivo.
We measured the magnitude of cutaneous responses to i.d. injections of C3a or C5a, and the extent of IgE-dependent mast cell degranulation and PCA responses, in mice containing mast cells that did or did not express C3aR or C5aR.
The majority of the skin swelling induced by i.d. injection of C3a or C5a required that mast cells at the site expressed C3aR or C5aR, respectively, and the extent of IgE-dependent degranulation of skin mast cells and of IgE-dependent PCA were significantly reduced when mast cells lacked either C3aR or C5aR. IgE-dependent PCA responses associated with local elevation of C3a occurred in antibody-deficient mice but not in mice deficient in FcεRIγ.
Expression of C3aR and C5aR by skin mast cells contributes importantly to the ability of C3a and C5a to induce skin swelling and can enhance mast cell degranulation and inflammation during IgE-dependent PCA in vivo.
Anaphylatoxin; anaphylaxis; C3a; C5a; complement; IgE; inflammation; mast cells
TH17 responses have recently been implicated to play a role in allergic airway diseases, but their local expression in the setting of allergic rhinitis (AR) and their regulation in allergic airway diseases remain unclear.
We sought to investigate the regulatory role of Clara cell 10-kDa protein (CC10), an endogenous regulator of airway inflammation, on TH17 responses in the setting of AR.
Wild-type and homozygous CC10-null mice were used to establish an ovalbumin (OVA)–induced AR model. Human recombinant CC10 was given during sensitization or challenge. TH17 responses in human subjects and mice were examined by using flow cytometry, quantitative RT-PCR assay, immunohistochemistry, and ELISA. The direct effect of CC10 on TH17 cells and CD11c+ dendritic cells (DCs) was studied by means of cell culture. Adoptive transfer was used to examine the influence of CC10-conditioned DCs on airway inflammation. The regulatory effect of CC10 on the expression of the CCL20 gene was tested by using the BEAS-2B cell line.
Compared with those of control subjects, TH17 responses were enhanced in the nasal mucosa of patients with AR. CC10-null mice with AR showed enhanced TH17 responses, and CC10 treatment significantly decreased TH17 responses. CC10 had no direct effect on in vitro TH17 cell differentiation. CC10 could significantly decrease the expression of OX40 ligand, IL-23, and IL-6 but enhance CD86 and TGF-β expression in DCs. Importantly, CC10 was able to inhibit TH17 cell polarization in the presence of OVA-pulsed DCs. CC10 pretreatment inhibited TH17 responses elicited by adoptive transfer of OVA-pulsed DCs. Furthermore, CC10 decreased the expression of CCL20 in BEAS-2B cells induced by inflammatory cytokines.
TH17 responses are enhanced in patients with AR, and CC10 inhibits TH17 responses through modulation of the function of DCs.
Allergic rhinitis; Clara cell 10-kDa protein; dendritic cell; inhibition; TH17 response
The skin of patients with atopic dermatitis (AD) has defects in keratinocyte differentiation, particularly in expression of the epidermal barrier protein filaggrin. AD skin lesions are often exacerbated by Staphylococcus aureus–mediated secretion of the virulence factor α-toxin. It is unknown whether lack of keratinocyte differentiation predisposes to enhanced lethality from staphylococcal toxins.
We investigated whether keratinocyte differentiation and filaggrin expression protect against cell death induced by staphylococcal α-toxin.
Filaggrin-deficient primary keratinocytes were generated through small interfering RNA gene knockdown. RNA expression was determined by using real-time PCR. Cell death was determined by using the lactate dehydrogenase assay. Keratinocyte cell survival in filaggrin-deficient (ft/ft) mouse skin biopsies was determined based on Keratin 5 staining. α-Toxin heptamer formation and acid sphingomyelinase expression were determined by means of immunoblotting.
We found that filaggrin expression, occurring as the result of keratinocyte differentiation, significantly inhibits staphylococcal α-toxin–mediated pathogenicity. Furthermore, filaggrin plays a crucial role in protecting cells by mediating the secretion of sphingomyelinase, an enzyme that reduces the number of α-toxin binding sites on the keratinocyte surface. Finally, we determined that sphingomyelinase enzymatic activity directly prevents α-toxin binding and protects keratinocytes against α-toxin–induced cytotoxicity.
The current study introduces the novel concept that S aureus α-toxin preferentially targets and destroys filaggrin-deficient keratinocytes. It also provides a mechanism to explain the increased propensity for S aureus–mediated exacerbation of AD skin disease.
Atopic dermatitis; skin barrier; filaggrin; acid sphingomyelinase; Staphylococcus aureus; α-toxin
The capacity of CD8+ T cells to control infections and mediate anti-tumor immunity requires the development and survival of effector and memory cells. IL-21 has emerged as a potent inducer of CD8+ T cell effector function and memory development in mouse models of infectious disease. However, the role of IL-21 and associated signaling pathways in protective CD8+ T cell immunity in humans is unknown.
To determine which signaling pathways mediate the effects of IL-21 on human CD8+ T cells and whether defects in these pathways contribute to disease pathogenesis in primary immunodeficiencies caused by mutations in components of the IL-21 signaling cascade.
Human primary immunodeficiencies resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Lymphocytes from patients with loss-of-function mutations in STAT1, STAT3 or IL21R were used to assess the respective roles of these genes in human CD8+ T cell differentiation in vivo and in vitro.
Mutations in STAT3 and IL21R, but not STAT1, lead to a decrease in multiple memory CD8+ T cell subsets in vivo, indicating that STAT3 signaling – possibly downstream of IL-21R - regulates the memory cell pool. Furthermore, STAT3 was important for inducing the lytic machinery in IL-21-stimulated naïve CD8+ T cells. However, this defect was overcome by TCR engagement.
The IL-21R/STAT3 pathway is required for many aspects of human CD8+ T cell behavior but in some cases can be compensated by other signals. This helps explain the relatively mild susceptibility to viral disease observed in STAT3 and IL-21R-deficient individuals.
autosomal dominant hyper-IgE syndrome; STAT3; STAT1; IL-21; human CD8+ T cells; memory; differentiation
Alternative splicing is important for increasing the complexity of the human proteome from a limited genome. Previous studies have shown that for some autoantigens, there is differential immunogenicity among alternatively spliced isoforms.
Herein, we tested the hypothesis that alternative splicing is a common feature for transcripts of autologous proteins that are autoantigens. The corollary hypothesis tested was that nonautoantigen transcripts have a lower frequency of alternative splicing.
The extent of alternative splicing within 45 randomly selected self-proteins associated with autoimmune diseases was compared with 9554 randomly selected proteins in the human genome by using bioinformatics analyses. Isoform-specific regions that resulted from alternative splicing were studied for their potential to be epitopes for antibodies or T-cell receptors.
Alternative splicing occurred in 100% of the autoantigen transcripts. This was significantly higher than the approximately 42% rate of alternative splicing observed in the 9554 randomly selected human gene transcripts (P < .001). Within the isoform-specific regions of the autoantigens, 92% and 88% encoded MHC class I and class II–restricted T-cell antigen epitopes, respectively, and 70% encoded antibody binding domains. Furthermore, 80% of the autoantigen transcripts underwent noncanonical alternative splicing, which is also significantly higher than the less than 1% rate in randomly selected gene transcripts (P < .001).
These studies suggest that noncanonical alternative splicing may be an important mechanism for the generation of untolerized epitopes that may lead to autoimmunity.
Furthermore, the product of a transcript that does not undergo alternative splicing is unlikely to be a target antigen in autoimmunity.
Autoantigens; alternative splicing; exons; isoforms; antigen epitopes; immunogenicity; immune tolerance; autoimmune diseases