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Particulate matter (PM) pollutant exposure, which induces oxidative stress and inflammation, and vitamin D insufficiency, which compromises immune regulation, are detrimental in asthma. Mechanistic cell culture experiments were undertaken to ascertain whether vitamin D abrogates PM-induced inflammatory responses of human bronchial epithelial cells (HBECs) through enhancement of antioxidant pathways. Transcriptome analysis, PCR and ELISA were undertaken to delineate markers of inflammation and oxidative stress; with comparison of expression in primary HBECs from healthy and asthmatic donors cultured with reference urban PM in the presence/absence of vitamin D. Transcriptome analysis identified over 500 genes significantly perturbed by PM-stimulation, including multiple pro-inflammatory cytokines. Vitamin D altered expression of a subset of these PM-induced genes, including suppressing IL6. Addition of vitamin D suppressed PM-stimulated IL-6 production, although to significantly greater extent in healthy versus asthmatic donor cultures. Vitamin D also differentially affected PM-stimulated GM-CSF, with suppression in healthy HBECs and enhancement in asthmatic cultures. Vitamin D increased HBEC expression of the antioxidant pathway gene G6PD, increased the ratio of reduced to oxidised glutathione, and in PM-stimulated cultures decreased the formation of 8-isoprostane. Pre-treatment with vitamin D decreased CXCL8 and further decreased IL-6 production in PM-stimulated cultures, an effect abrogated by inhibition of G6PD with DHEA, supporting a role for this pathway in the anti-inflammatory actions of vitamin D. In a study using HBECs from 18 donors, vitamin D enhanced HBEC antioxidant responses and modulated the immune response to PM, suggesting that vitamin D may protect the airways from pathological pollution-induced inflammation.

ObjectiveDysregulated immune responses are the cause of IBDs. Studies in mice and humans suggest a central role of interleukin (IL)-23-producing mononuclear phagocytes in disease pathogenesis. Mechanistic insights into the regulation of IL-23 are prerequisite for selective IL-23 targeting therapies as part of personalised medicine.DesignWe performed transcriptomic analysis to investigate IL-23 expression in human mononuclear phagocytes and peripheral blood mononuclear cells. We investigated the regulation of IL-23 expression and used single-cell RNA sequencing to derive a transcriptomic signature of hyperinflammatory monocytes. Using gene network correlation analysis, we deconvolved this signature into components associated with homeostasis and inflammation in patient biopsy samples.ResultsWe characterised monocyte subsets of healthy individuals and patients with IBD that express IL-23. We identified autosensing and paracrine sensing of IL-1α/IL-1β and IL-10 as key cytokines that control IL-23-producing monocytes. Whereas Mendelian genetic defects in IL-10 receptor signalling induced IL-23 secretion after lipopolysaccharide stimulation, whole bacteria exposure induced IL-23 production in controls via acquired IL-10 signalling resistance. We found a transcriptional signature of IL-23-producing inflammatory monocytes that predicted both disease and resistance to antitumour necrosis factor (TNF) therapy and differentiated that from an IL-23-associated lymphocyte differentiation signature that was present in homeostasis and in disease.ConclusionOur work identifies IL-10 and IL-1 as critical regulators of monocyte IL-23 production. We differentiate homeostatic IL-23 production from hyperinflammation-associated IL-23 production in patients with severe ulcerating active Crohn’s disease and anti-TNF treatment non-responsiveness. Altogether, we identify subgroups of patients with IBD that might benefit from IL-23p19 and/or IL-1α/IL-1β-targeting therapies upstream of IL-23.

The GP130 cytokine receptor subunit encoded by IL6ST is the shared receptor for ten cytokines of the IL-6 family. We describe a homozygous non-synonymous variant in IL6ST (p.R281Q) in a patient with craniosynostosis and retained deciduous teeth. We characterize the impact of the variant on cytokine signaling in vitro using transfected cell lines as well as primary patient-derived cells and support these findings using a mouse model with the corresponding genome-edited variant Il6st p.R279Q. We show that human GP130 p.R281Q is associated with selective loss of IL-11 signaling without affecting IL-6, IL-27, OSM, LIF, CT1, CLC, and CNTF signaling. In mice Il6st p.R279Q lowers litter size and causes facial synostosis and teeth abnormalities. The effect on IL-11 signaling caused by the GP130 variant shows incomplete penetrance but phenocopies aspects of IL11RA deficiency in humans and mice. Our data show that a genetic variant in a pleiotropic cytokine receptor can have remarkably selective defects.

Genetic variants in IL6ST encoding the shared cytokine receptor for the IL-6 cytokine family GP130 have been associated with a diverse number of clinical phenotypes and disorders. We provide a molecular classification for 59 reported rare IL6ST pathogenic or likely pathogenic variants and additional polymorphisms. Based on loss- or gain-of-function, cytokine selectivity, mono- and biallelic associations, and variable cellular mosaicism, we grade six classes of IL6ST variants and explore the potential for additional variants. We classify variants according to the American College of Medical Genetics and Genomics criteria. Loss-of-function variants with (i) biallelic complete loss of GP130 function that presents with extended Stüve-Wiedemann Syndrome; (ii) autosomal recessive hyper-IgE syndrome (HIES) caused by biallelic; and (iii) autosomal dominant HIES caused by monoallelic IL6ST variants both causing selective IL-6 and IL-11 cytokine loss-of-function defects; (iv) a biallelic cytokine-specific variant that exclusively impairs IL-11 signaling, associated with craniosynostosis and tooth abnormalities; (v) somatic monoallelic mosaic constitutively active gain-of-function variants in hepatocytes that present with inflammatory hepatocellular adenoma; and (vi) mosaic constitutively active gain-of-function variants in hematopoietic and non-hematopoietic cells that are associated with an immune dysregulation syndrome. In addition to Mendelian IL6ST coding variants, there are common non-coding cis-acting variants that modify gene expression, which are associated with an increased risk of complex immune-mediated disorders and trans-acting variants that affect GP130 protein function. Our taxonomy highlights IL6ST as a gene with particularly strong functional and phenotypic diversity due to the combinatorial biology of the IL-6 cytokine family and predicts additional genotype-phenotype associations.

Identification of the causes of poor oral vaccine immunogenicity in low-income countries might lead to more effective vaccines. We measured mucosal and systemic immune parameters at the time of vaccination with oral poliovirus vaccine (OPV) in 292 Indian infants aged 6–11 months, including plasma cytokines, leukocyte counts, fecal biomarkers of environmental enteropathy and peripheral blood T-cell phenotype, focused on gut-homing regulatory CD4+ populations. We did not find a distinct immune phenotype associated with OPV immunogenicity, although viral pathogens were more prevalent in stool at the time of immunization among infants who failed to seroconvert (63.9% vs. 45.6%, p  = 0.002). Using a machine-learning approach, we could predict seroconversion a priori using immune parameters and infection status with a median 58% accuracy (cross-validation IQR: 50–69%) compared with 50% expected by chance. Better identification of immune predictors of OPV immunogenicity is likely to require sampling of mucosal tissue and improved oral poliovirus infection models.

The prevalence of vitamin D insufficiency and deficiency has increased markedly in recent decades to current epidemic levels (Hyppönen E, et al. Am J Clin Nutr 2007;85:860–868). In parallel, there has been an increase in the incidence of a range of immune-mediated conditions ranging from cancer to autoimmune and respiratory diseases, including chronic obstructive pulmonary disease and asthma (Holick MF. N Engl J Med 2007;357:266–281; Finklea et al. Adv Nutr 2011;2:244–253). There is also an association with increased respiratory infections, which are the most common cause of asthma exacerbations (Finklea et al. Adv Nutr 2011;2:244–253). Together, this has resulted in considerable interest in the therapeutic potential of vitamin D to prevent and improve treatment of asthma and other respiratory diseases. To this end, data from clinical trials involving supplementation with active vitamin D, or more commonly a precursor, are starting to emerge. This review considers mechanisms by which vitamin D may act on the immune system to dampen inappropriate inflammatory responses in the airway while also promoting tolerance and antimicrobial defense mechanisms that collectively maintain respiratory health.

Background: Particulate matter (PM) pollutant exposure, which induces oxidative stress and inflammation, and vitamin D insufficiency, which compromises immune regulation, are detrimental in asthma. Objectives: Mechanistic cell culture experiments were undertaken to ascertain whether vitamin D abrogates PM-induced inflammatory responses of human bronchial epithelial cells (HBECs) through enhancement of antioxidant pathways. Methods: Transcriptome analysis, PCR and ELISA were undertaken to delineate markers of inflammation and oxidative stress; with comparison of expression in primary HBECs from healthy and asthmatic donors cultured with reference urban PM in the presence/absence of vitamin D. Results: Transcriptome analysis identified over 500 genes significantly perturbed by PM-stimulation, including multiple pro-inflammatory cytokines. Vitamin D altered expression of a subset of these PM-induced genes, including suppressing IL6. Addition of vitamin D suppressed PM-stimulated IL-6 production, although to significantly greater extent in healthy versus asthmatic donor cultures. Vitamin D also differentially affected PM-stimulated GM-CSF, with suppression in healthy HBECs and enhancement in asthmatic cultures. Vitamin D increased HBEC expression of the antioxidant pathway gene G6PD, increased the ratio of reduced to oxidised glutathione, and in PM-stimulated cultures decreased the formation of 8-isoprostane. Pre-treatment with vitamin D decreased CXCL8 and further decreased IL-6 production in PM-stimulated cultures, an effect abrogated by inhibition of G6PD with DHEA, supporting a role for this pathway in the anti-inflammatory actions of vitamin D. Conclusions: In a study using HBECs from 18 donors, vitamin D enhanced HBEC antioxidant responses and modulated the immune response to PM, suggesting that vitamin D may protect the airways from pathological pollution-induced inflammation.

Alpha-1 antitrypsin (AAT) is the most abundant serine protease inhibitor in the circulation, and is crucially protective in controlling pulmonary proteases including neutrophil elastase. AAT deficiency (AATD) is strongly associated with chronic obstructive pulmonary disease (COPD), primarily due to protease-antiprotease imbalance. However, recent studies also suggest immunomodulatory properties of AAT including the induction of IL-10 by dendritic cells and monocytes. Our group recently demonstrated that SERPINA1, the gene encoding AAT, is one of the most highly upregulated genes in vitamin D treated CD4+ T cells, and that AAT also upregulates IL-10 synthesis by these cells, in a complement C3a dependent manner. CD8+ T cells are implicated in the pathogenesis of COPD. Therefore the initial aim was to assess whether vitamin D increases AAT synthesis in CD8+ T cells. Vitamin D significantly upregulated SERPINA1/AAT in CD4+ T cells, with a much smaller effect in CD8+ T cells. Addition of exogenous TGF-β, previously shown to increase AAT secretion by other cell types, dose-dependently enhanced vitamin D induction of AAT in CD8+, but not CD4+, T cells. TGF-β with vitamin D increased VDR and RXRα in CD8+ T cells, whilst neutralization of TGF-β reduced the capacity of vitamin D to enhance both SERPINA1 and VDR in CD4+ T cells. These data imply that TGF-β modifies the response of vitamin D to human T cells. Data from a TGF-β bioassay suggested no difference in baseline secretion of bioactive TGF-β between the 2 cell types, but increased secretion by CD4+ T cells following vitamin D exposure, providing a potential explanation for the requirement of exogenous TGF-β in CD8+ T cell cultures. The next aim was to study AAT functions in T cells. Firstly, effects of wild-type (MM) and mutant (ZZ) AAT on CD8+ T cell immuno-regulatory function were assessed. However, little effect on cytokine responses including IL-10 induction was observed. As a result, gene expression microarrays were performed to further investigate AAT functions. These highlighted a low number of genes/pathways regulated by wild-type AAT, suggesting that AAT synthesised by CD8+ T cells may act on accessory cells, and/or that effects on CD8+ T cells may be indirect, requiring the presence of accessory cells in culture. The potential of vitamin D to increase AAT levels in AATD patients carrying the PiZZ mutation was next evaluated. The capacity of vitamin D +/- TGF-β to induce SERPINA1/AAT was compared in T cells from PiZZ patients, healthy control subjects and non-AATD COPD patients. The previously observed SERPINA1 induction by vitamin D +/- TGF-β in healthy individuals was reduced in PiZZ CD8+ but not CD4+ T cells. However, this response was significantly impaired at the protein level in both PiZZ CD4+ and CD8+ T cells, suggesting that T cells from AATD patients may exhibit a secretion defect. The IL-17 family cytokines are over-produced in severe and chronic respiratory disease. Since AAT may reduce IL-17A in animal models, evidence of dysregulated cytokine responses in PBMC cultures, following T cell receptor ligation, from AATD patients was investigated. Strikingly, an increased frequency of cells co-expressing IL-17A, with IL-17F and IL-22 was observed in PBMC cultures from PiZZ patients, in comparison to healthy individuals or older COPD patients. There was no difference in Th1 or Th2 cytokines including IFN-γ and IL-13 or IL-10 between the groups. Vitamin D increased IL-13 and decreased IFN-γ, IL-17A, IL-17F and IL-22. This enhanced Th17 response may be explained by the lack of AAT, although direct suppression by vitamin D is also possible, and this warrants further study. These findings suggest an interaction between vitamin D and AAT and support the concept that both are important mediators in the maintenance of respiratory health. Understanding how those mediators exert protective effects in the airways is critical in optimizing therapeutic strategies for AATD, where the only approved therapy has many limitations.

Identification of the causes of poor oral vaccine immunogenicity in low-income countries might lead to more effective vaccines. We measured mucosal and systemic immune parameters at the time of vaccination with oral poliovirus vaccine (OPV) in 292 Indian infants aged 6-11 months, including plasma cytokines, leukocyte counts, fecal biomarkers of environmental enteropathy and peripheral blood T-cell phenotype, focused on gut-homing regulatory CD4+ populations. We did not find a distinct immune phenotype associated with OPV immunogenicity, although viral pathogens were more prevalent in stool at the time of immunization among infants who failed to seroconvert (63.9% vs. 45.6%, p = 0.002). Using a machine-learning approach, we could predict seroconversion a priori using immune parameters and infection status with a median 58% accuracy (cross-validation IQR: 50-69%) compared with 50% expected by chance. Better identification of immune predictors of OPV immunogenicity is likely to require sampling of mucosal tissue and improved oral poliovirus infection models.

Melanoma is the deadliest type of skin cancer. CD20＋ melanoma stem cells （CSCs） are pivotal for metastasis and initiation of melanoma. Therefore, selective elimination of CD20＋ melanoma CSCs represents an effective treatment to eradicate melanoma. Salinomycin has emerged as an effective drug toward various CSCs. Due to its poor solubility, its therapeutic efficacy against melanoma CSCs has never been evaluated. In order to target CD20＋ melanoma CSCs, we designed salinomycin-loaded lipid-polymer nanoparticles with anti-CD20 aptamers （CD20-SA-NPs）. Using a single-step nanoprecipitation method, salinomycin-loaded lipid- polymer nanoparticles （SA-NPs） were prepared, then CD20-SA-NPs were obtained through conjugation of thiolated anti-CD20 aptamers to SA-NPs via a maleimide-thiol reaction. CD20-SA-NPs displayed a small size of 96.3 nm, encapsulation efficiency higher than 60% and sustained drug release ability. The uptake of CD20-SA-NPs by CD20＋ melanoma CSCs was significantly higher than that of SA-NPs and salinomycin, leading to greatly enhanced cytotoxic effects in vitro, thus the ICso values of CD20-SA-NPs were reduced to 5.7 and 2.6 pg/mL in A375 CD20＋ cells and WM266-4 CD＋ cells, respectively. CD20-SA-NPs showed a selective cytotoxicity toward CD20＋ melanoma CSCs, as evidenced by the best therapeutic efficacy in suppressing＊the formation of tumor spheres and the proportion of CD20＋ cells in melanoma cell lines. In mice bearing melanoma xenografts, administration of CD20-SA-NPs （salinomycin 5 mg·kg^-1·d^-1, iv, for 60 d） showed a superior efficacy in inhibition of melanoma growth compared with SA-NPs and salinomycin. In conclusion, CD20 is a superior target for delivering drugs to melanoma CSCs. CD20-SA-NPs display effective delivery of salinomycin to CD20＋ melanoma CSCs and represent a promising treatment for melanoma.