Exposure to isocyanates predicts atopic dermatitis prevalence and disrupts therapeutic pathways in commensal bacteria.

Atopic dermatitis (AD) is a chronic inflammatory skin condition increasing in industrial nations at a pace that suggests environmental drivers. We hypothesize that the dysbiosis associated with AD may signal microbial adaptations to modern pollutants. Having previously modeled the benefits of health-associated Roseomonas mucosa, we now show that R. mucosa fixes nitrogen in the production of protective glycerolipids and their ceramide by-products. Screening EPA databases against the clinical visit rates identified diisocyanates as the strongest predictor of AD. Diisocyanates disrupted the production of beneficial lipids and therapeutic modeling for isolates of R. mucosa as well as commensal Staphylococcus. Last, while topical R. mucosa failed to meet commercial end points in a placebo-controlled trial, the subgroup who completed the full protocol demonstrated sustained, clinically modest, but statistically significant clinical improvements that differed by study site diisocyanate levels. Therefore, diisocyanates show temporospatial and epidemiological association with AD while also inducing eczematous dysbiosis.

Assessing microbial manipulation and environmental pollutants in the pathogenesis of psoriasis.

The cutaneous microbiome is increasingly recognized as a contributor to skin diseases like atopic dermatitis (AD) and psoriasis. Although traditionally AD and psoriasis have been viewed as having opposing immunologic findings, recent evidence suggests an overlap in ceramide-family lipid production in the protection against symptoms. We recently identified that specific environmental pollutants may drive dysbiosis through direct suppression of ceramide-family lipids produced by health-associated skin bacteria in atopic dermatitis (AD). We further demonstrated that one such bacteria, Roseomonas mucosa, generated significant clinical improvement in AD lasting beyond active treatment via lipid-mediated modulation of tumor necrosis factor (TNF) signaling. To assess the potential preclinical benefit of R. mucosa in psoriasis we assessed for direct effects on surface TNF signaling in cell cultures and identified direct effects on the TNF axis. We also identified preclinical efficacy of R. mucosa treatment in the imiquimod mouse model of psoriasis. Finally, we expanded our previous environmental assessment for psoriasis to include more traditional markers of air quality and found a strong association between disease rates and ambient carbon monoxide (CO), nitrogen dioxide (NO2), and particulate matter (PM). At the current stage this work is speculative but does support consideration of further preclinical models and/or clinical assessments to evaluate any potential for therapeutic benefit through microbial manipulation and/or environmental mitigation.

Therapeutic candidates for keloid scars identified by qualitative review of scratch assay research for wound healing.

The scratch assay is an in vitro technique used to analyze cell migration, proliferation, and cell-to-cell interaction. In the assay, cells are grown to confluence and then 'scratched' with a sterile instrument. For the cells in the leading edge, the resulting polarity induces migration and proliferation in attempt to 'heal' the modeled wound. Keloid scars are known to have an accelerated wound closure phenotype in the scratch assay, representing an overactivation of wound healing. We performed a qualitative review of the recent literature searching for inhibitors of scratch assay activity that were already available in topical formulations under the hypothesis that such compounds may offer therapeutic potential in keloid treatment. Although several shortcomings in the scratch assay literature were identified, caffeine and allicin successfully inhibited the scratch assay closure and inflammatory abnormalities in the commercially available keloid fibroblast cell line. Caffeine and allicin also impacted ATP production in keloid cells, most notably with inhibition of non-mitochondrial oxygen consumption. The traditional Chinese medicine, shikonin, was also successful in inhibiting scratch closure but displayed less dramatic impacts on metabolism. Together, our results partially summarize the strengths and limitations of current scratch assay literature and suggest clinical assessment of the therapeutic potential for these identified compounds against keloid scars may be warranted.

Analysis of the mechanism of damage produced by thiazole orange photoinactivation in apheresis platelets.

BACKGROUND: Pathogen Reduction Technologies (PRTs) are broad spectrum nucleic acid replication-blocking antimicrobial treatments designed to mitigate risk of infection from blood product transfusions. Thiazole Orange (TO), a photosensitizing nucleic acid dye, was previously shown to photoinactivate several types of bacterial and viral pathogens in RBC suspensions without adverse effects on function. In this report we extended TO treatment to platelet concentrates (PCs) to see whether it is compatible with in vitro platelet functions also, and thus, could serve as a candidate technology for further evaluation. MATERIAL AND METHODS: PCs were treated with TO, and an effective treatment dose for inactivation of Staphylococci was identified. Platelet function and physiology were then evaluated by various assays in vitro. RESULTS: Phototreatment of PCs yielded significant reduction (≥4-log) in Staphylococci at TO concentrations ≥20 µM. However, treatment with TO reduced aggregation response to collagen over time, and platelets became unresponsive by 24 hours post-treatment (from >80% at 1 h to 0% at 24 h). TO treatment also significantly increased CD62P expression (<1% CD62P+ for untreated and >50% for TO treated at 1 h) and induced apoptosis in platelets (<1% Annexin V+ for untreated and >50% for TO treated at 1 h) and damaged mitochondrial DNA. A mitochondria-targeted antioxidant and reactive oxygen species (ROS) scavenger Mito-Tempo mitigated these adverse effects. DISCUSSION: The results demonstrate that TO compromises mitochondria and perturbs internal signaling that activates platelets and triggers apoptosis. This study illustrates that protecting platelet mitochondria and its functions should be a fundamental consideration in selecting a PRT for transfusion units containing platelets, such as PCs.

Tumor Necrosis Factor Receptors: Pleiotropic Signaling Complexes and Their Differential Effects.

Since its discovery in 1975, TNFα has been a subject of intense study as it plays significant roles in both immunity and cancer. Such attention is well deserved as TNFα is unique in its engagement of pleiotropic signaling via its two receptors: TNFR1 and TNFR2. Extensive research has yielded mechanistic insights into how a single cytokine can provoke a disparate range of cellular responses, from proliferation and survival to apoptosis and necrosis. Understanding the intracellular signaling pathways induced by this single cytokine via its two receptors is key to further revelation of its exact functions in the many disease states and immune responses in which it plays a role. In this review, we describe the signaling complexes formed by TNFR1 and TNFR2 that lead to each potential cellular response, namely, canonical and non-canonical NF-κB activation, apoptosis and necrosis. This is followed by a discussion of data from in vivo mouse and human studies to examine the differential impacts of TNFR1 versus TNFR2 signaling.

A high-throughput inhibition assay to study MERS-CoV antibody interactions using image cytometry.

The emergence of new pathogens, such as Middle East respiratory syndrome coronavirus (MERS-CoV), poses serious challenges to global public health and highlights the urgent need for methods to rapidly identify and characterize potential therapeutic or prevention options, such as neutralizing antibodies. Spike (S) proteins are present on the surface of MERS-CoV virions and mediate viral entry. S is the primary target for MERS-CoV vaccine and antibody development, and it has become increasingly important to understand MERS-CoV antibody binding specificity and function. Commonly used serological methods like ELISA, biolayer interferometry, and flow cytometry are informative, but limited. Here, we demonstrate a high-throughput protein binding inhibition assay using image cytometry. The image cytometry-based high-throughput screening method was developed by selecting a cell type with high DPP4 expression and defining optimal seeding density and protein binding conditions. The ability of monoclonal antibodies to inhibit MERS-CoV S binding was then tested. Binding inhibition results were comparable with those described in previous literature for MERS-CoV spike monomer and showed similar patterns as neutralization results. The coefficient of variation (CV) of our cell-based assay was <10%. The proposed image cytometry method provides an efficient approach for characterizing potential therapeutic antibodies for combating MERS-CoV that compares favorably with current methods. The ability to rapidly determine direct antibody binding to host cells in a high-throughput manner can be applied to study other pathogen-antibody interactions and thus can impact future research on viral pathogens.

TNF overproduction impairs epithelial staphylococcal response in hyper IgE syndrome.

Autosomal dominant hyper IgE syndrome (AD-HIES), or Job's syndrome, is a primary immune deficiency caused by dominant-negative mutations in STAT3. Recurrent Staphylococcus aureus skin abscesses are a defining feature of this syndrome. A widely held hypothesis that defects in peripheral Th17 differentiation confer this susceptibility has never been directly evaluated. To assess the cutaneous immune response in AD-HIES, we induced suction blisters in healthy volunteers (HVs) and patients with AD-HIES and then challenged the wound with lethally irradiated bacteria. We show that cutaneous production of IL-17A and IL-17F was normal in patients with AD-HIES. Overproduction of TNF-α differentiated the responses in AD-HIES from HVs. This was associated with reduced IL-10 family signaling in blister-infiltrating cells and defective epithelial cell function. Mouse models of AD-HIES recapitulated these aberrant epithelial responses to S. aureus and involved defective epithelial-to-mesenchymal transition (EMT) rather than a failure of bacterial killing. Defective responses in mouse models of AD-HIES and primary keratinocyte cultures from patients with AD-HIES could be reversed by TNF-α blockade and by drugs with reported modulatory effects on EMT. Our results identify these as potential therapeutic approaches in patients with AD-HIES suffering S. aureus infections.

IL-20 Signaling in Activated Human Neutrophils Inhibits Neutrophil Migration and Function.

Neutrophils possess multiple antimicrobial mechanisms that are critical for protection of the host against infection with extracellular microbes, such as the bacterial pathogen Staphylococcus aureus Recruitment and activation of neutrophils at sites of infection are driven by cytokine and chemokine signals that directly target neutrophils via specific cell surface receptors. The IL-20 subfamily of cytokines has been reported to act at epithelial sites and contribute to psoriasis, wound healing, and anti-inflammatory effects during S. aureus infection. However, the ability of these cytokines to directly affect neutrophil function remains incompletely understood. In this article, we show that human neutrophils altered their expression of IL-20R chains upon migration and activation in vivo and in vitro. Such activation of neutrophils under conditions mimicking infection with S. aureus conferred responsiveness to IL-20 that manifested as modification of actin polymerization and inhibition of a broad range of actin-dependent functions, including phagocytosis, granule exocytosis, and migration. Consistent with the previously described homeostatic and anti-inflammatory properties of IL-20 on epithelial cells, the current study provides evidence that IL-20 directly targets and inhibits key inflammatory functions of neutrophils during infection with S. aureus.

Vaccine protection of leukopenic mice against Staphylococcus aureus bloodstream infection.

The risk for Staphylococcus aureus bloodstream infection (BSI) is increased in immunocompromised individuals, including patients with hematologic malignancy and/or chemotherapy. Due to the emergence of antibiotic-resistant strains, designated methicillin-resistant S. aureus (MRSA), staphylococcal BSI in cancer patients is associated with high mortality; however, neither a protective vaccine nor pathogen-specific immunotherapy is currently available. Here, we modeled staphylococcal BSI in leukopenic CD-1 mice that had been treated with cyclophosphamide, a drug for leukemia and lymphoma patients. Cyclophosphamide-treated mice were highly sensitive to S. aureus BSI and developed infectious lesions lacking immune cell infiltrates. Virulence factors of S. aureus that are key for disease establishment in immunocompetent hosts-α-hemolysin (Hla), iron-regulated surface determinants (IsdA and IsdB), coagulase (Coa), and von Willebrand factor binding protein (vWbp)-are dispensable for the pathogenesis of BSI in leukopenic mice. In contrast, sortase A mutants, which cannot assemble surface proteins, display delayed time to death and increased survival in this model. A vaccine with four surface antigens (ClfA, FnBPB, SdrD, and SpAKKAA), which was identified by genetic vaccinology using sortase A mutants, raised antigen-specific immune responses that protected leukopenic mice against staphylococcal BSI.