Clifton F. Hall

Cytokine Milieu of Atopic Dermatitis, as Compared to Psoriasis, Skin Prevents Induction of Innate Immune Response Genes

Atopic dermatitis (AD) and psoriasis are the two most common chronic skin diseases. However patients with AD, but not psoriasis, suffer from frequent skin infections. To understand the molecular basis for this phenomenon, skin biopsies from AD and psoriasis patients were analyzed using GeneChip microarrays. The expression of innate immune response genes, human β defensin (HBD)-2, IL-8, and inducible NO synthetase (iNOS) was found to be decreased in AD, as compared with psoriasis, skin (HBD-2, p = 0.00021; IL-8, p = 0.044; iNOS, p = 0.016). Decreased expression of the novel antimicrobial peptide, HBD-3, was demonstrated at the mRNA level by real-time PCR (p = 0.0002) and at the protein level by immunohistochemistry (p = 0.0005). By real-time PCR, our data confirmed that AD, as compared with psoriasis, is associated with elevated skin production of Th2 cytokines and low levels of proinflammatory cytokines such as TNF-α, IFN-γ, and IL-1β. Because HBD-2, IL-8, and iNOS are known to be inhibited by Th2 cytokines, we examined the effects of IL-4 and IL-13 on HBD-3 expression in keratinocyte culture in vitro. We found that IL-13 and IL-4 inhibited TNF-α- and IFN-γ-induced HBD-3 production. These studies indicate that decreased expression of a constellation of antimicrobial genes occurs as the result of local up-regulation of Th2 cytokines and the lack of elevated amounts of TNF-α and IFN-γ under inflammatory conditions in AD skin. These observations could explain the increased susceptibility of AD skin to microorganisms, and suggest a new fundamental rule that may explain the mechanism for frequent infection in other Th2 cytokine-mediated diseases.

The Effects of Airway Microbiome on Corticosteroid Responsiveness in Asthma

RATIONALE The role of airway microbiome in corticosteroid response in asthma is unknown. OBJECTIVES To examine airway microbiome composition in patients with corticosteroid-resistant (CR) asthma and compare it with patients with corticosteroid-sensitive (CS) asthma and normal control subjects and explore whether bacteria in the airways of subjects with asthma may direct alterations in cellular responses to corticosteroids. METHODS 16S rRNA gene sequencing was performed on bronchoalveolar lavage (BAL) samples of 39 subjects with asthma and 12 healthy control subjects. In subjects with asthma, corticosteroid responsiveness was characterized, BAL macrophages were stimulated with pathogenic versus commensal microorganisms, and analyzed by real-time polymerase chain reaction for the expression of corticosteroid-regulated genes and cellular p38 mitogen-activated protein kinase (MAPK) activation. MEASUREMENTS AND MAIN RESULTS Of the 39 subjects with asthma, 29 were CR and 10 were CS. BAL microbiome from subjects with CR and CS asthma did not differ in richness, evenness, diversity, and community composition at the phylum level, but did differ at the genus level, with distinct genus expansions in 14 subjects with CR asthma. Preincubation of asthmatic airway macrophages with Haemophilus parainfluenzae, a uniquely expanded potential pathogen found only in CR asthma airways, resulted in p38 MAPK activation, increased IL-8 (P < 0.01), mitogen-activated kinase phosphatase 1 mRNA (P < 0.01) expression, and inhibition of corticosteroid responses (P < 0.05). This was not observed after exposure to commensal bacterium Prevotella melaninogenica. Inhibition of transforming growth factor-β-associated kinase-1 (TAK1), upstream activator of MAPK, but not p38 MAPK restored cellular sensitivity to corticosteroids. CONCLUSIONS A subset of subjects with CR asthma demonstrates airway expansion of specific gram-negative bacteria, which trigger TAK1/MAPK activation and induce corticosteroid resistance. TAK1 inhibition restored cellular sensitivity to corticosteroids.

Corticosteroid-resistant asthma is associated with classical antimicrobial activation of airway macrophages

BACKGROUND The cause of corticosteroid-resistant (CR) asthma is unknown. OBJECTIVE We sought to perform gene microarray analyses by using bronchoalveolar lavage (BAL) cells from well-characterized subjects with CR asthma and subject with corticosteroid-sensitive (CS) asthma to elucidate the differential expression of genes that contribute to the development of corticosteroid resistance. METHODS The patients were characterized as having CR or CS asthma based on FEV(1) percent predicted improvement after a 1-week course of oral prednisone. Expression of selected gene targets was verified by means of real-time PCR and ELISA. RESULTS Microarray analyses demonstrated significantly higher levels (>3-fold increase, P < .05) of transcripts for TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, CXCL1, CXCL2, CXCL3, CXCL8 (IL-8), CCL3, CCL4, and CCL20 in BAL cells of subjects with CR asthma. These findings, confirmed by means of 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 (early growth response 1, dual-specificity phosphatase 2, molecule possessing ankyrin repeats induced by LPS, and TNF-alpha-induced protein 3) were significantly increased in BAL samples from subjects with CR asthma (P < .05). These patients had significantly higher amounts (1444.0 +/- 457.3 pg/mg total protein) of LPS in BAL fluid than seen in subjects with CS asthma (270.5 +/- 216.0 pg, P < .05), as detected by using the LAL assay and confirmed by means of gas chromatographic/mass spectrometric analysis. Prolonged exposure to LPS induced functional steroid resistance to dexamethasone in normal human monocytes, as demonstrated by persistently increased IL-6 levels in the presence of dexamethasone. CONCLUSIONS Classical macrophage activation and induction of LPS signaling pathways along with high endotoxin levels detected in BAL fluid from subjects with CR asthma suggest that LPS exposure might contribute to CR asthma.

Divergent expression and function of glucocorticoid receptor β in human monocytes and T cells

Glucocorticoid (GC) insensitivity is a significant problem in the treatment of immune‐mediated diseases. The current study examined whether T cells and monocytes differed in their response to GC and the potential molecular basis for their variation in response to steroids. Functional studies revealed that dexamethasone (DEX) inhibited phorbol 12‐myristate 13‐acetate/ionomycin‐induced tumor necrosis factor α and interleukin‐6 production to a significantly lesser extent in monocytes than T cells. In parallel, a significantly longer period of time was required for DEX to induce the steroid‐responsive gene, mitogen‐activated protein kinase phosphatase‐1 (MKP‐1), in human monocytes as compared with T cells. It is interesting that such differences were not observed between murine T cells and monocytes. GC receptor β (GCRβ) is a splicing variant of the classic GCR, GCRα, which functions as a dominant‐negative inhibitor of GCRα in humans, not mice (as mice do not express GCRβ mRNA as a result of a difference in the murine GCR 9b exon sequence). It was found that human monocytes had a significantly higher level of GCRβ than T cells. Furthermore, GCRβ was found in the cytoplasm and nucleus of monocytes, and GCRβ was localized to the nucleus of T cells. This raised the possibility that GCRβ in the cytoplasm could affect GCRα cellular shuttling in response to DEX. Indeed, we found that DEX‐induced nuclear translocation of GCRα was decreased in monocytes as compared with T cells. Specific RNA silencing of GCRβ in human monocytes resulted in enhanced steroid‐induced GCRα transactivation and transrepression. Our data suggest that GCRβ contributes to variation in the GC responses of monocytes versus T cells.

Inhibition of transcription factor specificity protein 1 alters the gene expression profile of keratinocytes leading to upregulation of kallikrein-related peptidases and thymic stromal lymphopoietin.

Transcription factor specificity protein 1 (Sp1) is involved in diverse cellular functions. We recently found that Sp1 was significantly decreased in skin biopsies from patients with atopic dermatitis (AD) and had an even greater reduction in AD patients with a history of eczema herpeticum. In the current study, we sought to better understand the role of Sp1 in skin biological processes by using a small interfering RNA (siRNA) technique to knock down Sp1 gene expression in normal human keratinocytes (NHK) and investigated the genome-wide gene expression profiling of Sp1- silenced NHK. The gene arrays revealed that 53 genes had more than three-fold changes in expression in Sp1-silenced NHK as compared to scrambled siRNA silenced cells. Strikingly, six kallikrein-related peptidase genes, KLK5, KLK6, KLK7, KLK8, KLK10, and KLK12 were up-regulated in NHK following Sp1 silencing. Functionally, protease activity was significantly enhanced in Sp1-silenced keratinocytes as compared to scrambled siRNA silenced keratinocytes. Moreover, thymic stromal lymphopoietin (TSLP), an epithelial derived TH2 promoting cytokine, was induced in Sp1-silenced keratinocytes due to elevated kallikrein activity. These results indicate that Sp1 expression deficiency leads to abnormally increased kallikrein protease activity in keratinocytes and may contribute to TH2 immune responses in the skin by inducing TSLP.

Lipid abnormalities in atopic skin are driven by type 2 cytokines

Lipids in the stratum corneum of atopic dermatitis (AD) patients differ substantially in composition from healthy subjects. We hypothesized that hyperactivated type 2 immune response alters AD skin lipid metabolism. We have analyzed stratum corneum lipids from nonlesional and lesional skin of AD subjects and IL-13 skin-specific Tg mice. We also directly examined the effects of IL-4/IL-13 on human keratinocytes in vitro. Mass spectrometric analysis of lesional stratum corneum from AD subjects and IL-13 Tg mice revealed an increased proportion of short-chain (N-14:0 to N-24:0) NS ceramides, sphingomyelins, and 14:0-22:0 lysophosphatidylcholines (14:0-22:0 LPC) with a simultaneous decline in the proportion of corresponding long-chain species (N-26:0 to N-32:0 sphingolipids and 24:0-30:0 LPC) when compared with healthy controls. An increase in short-chain LPC species was also observed in nonlesional AD skin. Similar changes were observed in IL-4/IL-13-driven responses in Ca2+-differentiated human keratinocytes in vitro, all being blocked by STAT6 silencing with siRNA. RNA sequencing analysis performed on stratum corneum of AD as compared with healthy subjects identified decreased expression of fatty acid elongases ELOVL3 and ELOVL6 that contributed to observed changes in atopic skin lipids. IL-4/IL-13 also inhibited ELOVL3 and ELOVL6 expression in keratinocyte cultures in a STAT6-dependent manner. Downregulation of ELOVL3/ELOVL6 expression in keratinocytes by siRNA decreased the proportion of long-chain fatty acids globally and in sphingolipids. Thus, our data strongly support the pathogenic role of type 2 immune activation in AD skin lipid metabolism.

Minimally invasive skin tape strip RNA sequencing identifies novel characteristics of the type 2–high atopic dermatitis disease endotype

Background: Expression profiling of skin biopsy specimens has established molecular features of the skin in patients with atopic dermatitis (AD). The invasiveness of biopsies has prevented their use in defining individual‐level AD pathobiological mechanisms (endotypes) in large research studies. Objective: We sought to determine whether minimally invasive skin tape strip transcriptome analysis identifies gene expression dysregulation in AD and molecular disease endotypes. Methods: We sampled nonlesional and lesional skin tape strips and biopsy specimens from white adult patients with AD (18 male and 12 female patients; age [mean ± SE], 36.3 ± 2.2 years) and healthy control subjects (9 male and 16 female subjects; age [mean ± SE], 34.8 ± 2.2 years). AmpliSeq whole‐transcriptome sequencing was performed on extracted RNA. Differential expression, clustering/pathway analyses, immunostaining of skin biopsy specimens, and clinical trait correlations were performed. Results: Skin tape expression profiles were distinct from skin biopsy profiles and better sampled epidermal differentiation complex genes. Skin tape expression of 29 immune and epidermis‐related genes (false discovery rate < 5%) separated patients with AD from healthy subjects. Agnostic gene set analyses and clustering revealed 50% of patients with AD exhibited a type 2 inflammatory signature (type 2–high endotype) characterized by differential expression of 656 genes, including overexpression of IL13, IL4R, CCL22, CCR4 (log2 fold change = 5.5, 2.0, 4.0, and 4.1, respectively) and at a pathway level by TH2/dendritic cell activation. Both expression and immunostaining of skin biopsy specimens indicated this type 2–high group was enriched for inflammatory, type 2–skewed dendritic cells expressing Fc&egr;RI. The type 2–high endotype group exhibited more severe disease by using both the Eczema Area and Severity Index score and body surface area covered by lesions. Conclusion: Minimally invasive expression profiling of nonlesional skin reveals stratification in AD molecular pathology by type 2 inflammation that correlates with disease severity.

Th2 Cytokines Suppress Lipoteichoic Acid Induced Matrix Metalloproteinase Expression and Keratinocyte Migration in Response to Wounding

Recent clinical trials have established a central role for the Th2 cytokines IL-4 and IL-13 in the pathology of atopic dermatitis (AD) (Beck et al., 2014). Although blockade of the IL-4 and IL-13 receptors causes significant clearing of skin lesions, a direct effect of Th2 cytokines on wound healing processes has not yet been demonstrated. Furthermore, AD patients are frequently affected by infection with the pathogen, Staphylococcus aureus (Boguniewicz and Leung, 2011). Elevated levels of staphylococcal products are frequently found on the skin of affected patients (Travers et al., 2010), and these products may affect the wound healing process as well.